Molnupiravir and Its Active Form, EIDD-1931, Show Potent Antiviral Activity against Enterovirus Infections In Vitro and In Vivo

Enterovirus infections can cause hand, foot, and mouth disease (HFDM), aseptic meningitis, encephalitis, myocarditis, and acute flaccid myelitis, leading to death of infants and young children. However, no specific antiviral drug is currently available for the treatment of this type of infection. The Unites States and United Kingdom health authorities recently approved a new antiviral drug, molnupiravir, for the treatment of COVID-19. In this study, we reported that molnupiravir (EIDD-2801) and its active form, EIDD-1931, have broad-spectrum anti-enterovirus potential. Our data showed that EIDD-1931 could significantly reduce the production of EV-A71 progeny virus and the expression of EV-A71 viral protein at non-cytotoxic concentrations. The results of the time-of-addition assay suggest that EIDD-1931 acts at the post-entry step, which is in accordance with its antiviral mechanism. The intraperitoneal administration of EIDD-1931 and EIDD-2801 protected 1-day-old ICR suckling mice from lethal EV-A71 challenge by reducing the viral load in various tissues of the infected mice. The pharmacokinetics analysis indicated that the plasma drug concentration overwhelmed the EC50 for enteroviruses, suggesting the clinical potential of molnupiravir against enteroviruses. Thus, molnupiravir along with its active form, EIDD-1931, may be a promising drug candidate against enterovirus infections.


Introduction
Enterovirus belongs to the Enterovirus genus of the Picornaviridae family. Enterovirus infections cause hand, foot, and mouth disease (HFMD), myocarditis, and a series of neurological complications in infants and young children worldwide [1,2]. Enteroviruses include polioviruses, echoviruses, coxsackieviruses, and numbered enteroviruses [3]. Among these species, enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), and coxsackievirus A16 (CV-A16) have the potential to cause fatal infections, including aseptic meningitis (AM) and encephalitis [4,5]. Enterovirus D68 (EV-D68) sometimes causes severe neurological complications, such as acute flaccid myelitis (AFM) [6]. Coxsackievirus B3 (CV-B3), a cardiotropic virus, has been identified as one of the leading causes of viral myocarditis [7][8][9]. Although most enterovirus infections cause only mild and self-limiting diseases, the large number of cases and high prevalence of enterovirus infections throughout the world highlight the need for specific antiviral drugs against enteroviruses [10][11][12][13][14]. Unfortunately, there are no antiviral drugs currently approved to treat enterovirus infections. Although three inactivated monovalent EV-A71 vaccines have been widely used in the prevention of hand, foot, and mouth disease (HFDM) and some clinical trials have reported that these vaccines can provide efficient protection against EV-A71-associated HFMD, a cross-protection effect against CV-A6, CV-A10, and CV-A16 has rarely been observed [15][16][17][18]. Due to the vast number of different enteroviral serotypes, research on individual vaccines against all types of enteroviruses is not feasible. Therefore, the development of broad-spectrum antiviral drugs with activity against multiple serotypes of enteroviruses is urgently needed.
β-D-N 4 -hydroxycytidine (EIDD-1931, the active form of Molnupiravir) was designed and synthesized as a ribonucleotide analog and is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of lethal mutagenesis and thereby preventing the generation of offspring viruses [19]. EIDD-1931 was found capable of inhibiting various RNA viruses, including hepatitis C virus, influenza viruses, respiratory syncytial virus, Ebola virus, Venezuelan equine encephalitis virus, SARS-CoV-2, and human seasonal coronaviruses [19][20][21][22][23]. As a ribonucleoside analog, EIDD-1931 is quickly metabolized in the enterocytes of non-human primates after oral administration, which could reflect poor human bioavailability. To overcome this problem, EIDD-2801 (molnupiravir, prodrug of EIDD-1931) was designed. In vitro experiments demonstrated that EIDD-2801 increased oral bioavailability in non-human primates and ferrets compared to EIDD-1931 [24].
In this study, we evaluated the antiviral activity of EIDD-1931 and EIDD-2801 against enterovirus EV-A71 in vivo and in vitro, verified the stage of action of EIDD-1931 against EV-A71, and measured the broad-spectrum anti-enterovirus activities of EIDD-1931 and EIDD-2801.
EIDD-1931(cat no. T8498) and EIDD-2801 (cat no. T8309) powders were purchased from TargetMol (Shanghai, China), and NITD008 (7-Deaza-2 -C-acetylene-adenosine, cat no. HY-12957) powders were purchased from MCE (Shanghai, China). They were dissolved in DMSO at a 100 mM stock concentration and then stored at −20 ºC for in vitro tests. For the in vivo activity evaluation, EIDD-1931 and EIDD-2801 were solubilized in saline and then subjected to alternating ultrasound treatment to ensure full dissolution.

Antiviral Activity Assay
The antiviral activity assay was performed in 96-well plates as previously described [31]. Briefly, RD, Vero, and Huh7 cells were seeded in a white-walled clear-bottom 96-well plate at 1.0 × 10 4 cells/well and grown for 24 h before infection. EIDD-1931 or EIDD-2801 was added to the cells in a 3-fold dilution series (ranging from 200 to 0.01 µM) with 2% FBS DMEM, and DMSO treatment was set as a control. The virus was diluted to 100× TCID 50 using the 2% FBS DMEM and added to the above-mentioned 96-well plates. After incubation at 37 • C for 72 h, the antiviral effects of the test compounds were measured using a CellTiter-Glo cell viability assay kit (Promega, cat no. G7570) following the manufacturer's instructions. The luminescence was read using a SpectraMax M5 microplate reader (Molecular Devices). The half-maximal effective concentration (EC 50 ) was calculated using Origin 9.0 software.
To explore the inhibitory activity of EIDD-1931 and EIDD-2801 on EV-A71, the viral loads in the supernatant and the viral RNA levels in the cell lysates were measured by TCID 50 and quantitative real-time PCR (qRT-PCR), respectively. RD cells were seeded in 12-well plates at 4.0 × 10 5 cells/well. After 24 h, the cells were incubated with the EV-A71 virus at a multiplicity of infection (MOI) of 0.1 PFU/mL and the diluted test compounds. After 1.5 h, the supernatant was aspirated and supplemented with 1 mL of 2% FBS DMEM containing different concentrations of test compounds. The virus particle yields in the supernatant and viral RNA in cells were quantified at 30 h post infection (h.p.i.).

Immunofluorescence Assay
Vero cells were seeded in 96-well plates at 1.0 × 10 4 cells/well and grown for 24 h before infection, followed by infection with the EV-A71 H strain at an MOI of 1. The test compounds were added at the indicated concentrations. After 16 h, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed with PBS. After that, the cells were perforated by 0.1% Triton X-100 at room temperature for 30 min and blocked by 5% BSA at 37 • C for 30 min, followed by incubation with mouse anti-EV-A71 VP1 (12D7) antibody (1:5000) at 37 • C for 1 h and with Alexa Fluor 546 donkey anti-mouse IgG (H + L) antibody (1:500) for 1 h at 37 • C. The cells were washed three times, and the nucleus was stained using the Hoechst 33,342 fluorescent stain for 30 min at room temperature. The cell images were captured using a Leica DMi8 inverted microscope (Leica).

Time-of-Drug-Addition Assay
Time-of-drug-addition assay was carried out according to previous reports [32]. Briefly, RD cells were plated in 12-well plates at 4.0 × 10 5 cells/well and incubated overnight. Then, the cells were treated with the EV-A71 H strain at an MOI of 0.01 PFU/mL and EIDD-1931 (10 µM) or NITD008 (1 µM) according to the drug addition schedule. At 30 h.p.i., the cells were harvested, and total RNA was extracted then subjected to qRT-PCR.

Quantitative Real-Time PCR
Total cellular RNA was extracted from the cell samples using an RNeasy mini kit (Qiagen, Hilden, Germany, cat no. 74106). Quantitative real-time PCR (qRT-PCR) was performed using the One Step PrimeScript RT-PCR Kit (TaKaRa, Otsu, Japan, cat no. RR064A) according to the manufacturer's instructions. The details of the primer and probe sequences specific to the EV-A71 virus are as follows: forward primer, 5 -CCAATCTCAGCGGCTTGG AG-3 ; reverse primer, 5 -CACTCAAGCTCTACCGGCAC-3 ; and probe, FAM-TCCAATCG ATGGCTGCTCACCTGCGT-BHQ1. Virus RNA copy numbers were calculated by comparing the cycle threshold (Ct) value obtained from each sample with that of the standard curve based on known copy numbers.

Western Blot Assay
RD cells were seeded in 12-well plates at 4.0 × 10 5 cells/well, incubated overnight, and then supplemented with 1 mL of 2% FBS DMEM containing EV-A71 virus at an MOI of 0.1 and the test compound (EIDD-1931: ranging from 10 to 0.63 µM; EIDD-2801: ranging from 100 to 6.3 µM). The cell control group was supplemented with 1 mL of 2% FBS DMEM and incubated for 1.5 h at 37 • C; the cells were then washed three times with PBS and supplemented with 1 mL of 2% FBS DMEM. After 24 h, the cells were washed three times with cold PBS, followed by addition of 150 µL of RIPA lysis buffer (APPLYGEN, cat no. 1053) containing protease and phosphatase inhibitor cocktail (Thermo, cat no. A32961). The proteins were separated on a 10% SDS-PAGE gel and then transferred onto a PVDF membrane. The PVDF membranes were blocked using 5% skim milk for 1 h at room temperature and then incubated with anti-EV-A71 VP1 antibody 12D7 (1:1000) or anti-alpha tubulin antibody (1:5000) as a control at 4 • C overnight. The membranes were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5000) at room temperature for 1 h. Finally, the protein bands were imaged using a fluorchem imaging system (ProteinSimple).

EV-A71 Infection in Mice
The in vivo therapeutic efficacy of EIDD-1931 and EIDD-2801 was evaluated using 1-day-old suckling ICR mice as previously described [30]. Briefly, groups of 1-day-old suckling mice (purchased from the Beijing Vital River Laboratory, China) were intraperitoneally (i.p.) inoculated with 25 µL of the EV-A71 H strain (10 6 PFU per mouse). After 4 h post infection, EIDD-1931 (200, 66, 22, and 7.41 mg/kg) and EIDD-2801 (200, 66, 22, and 7.41 mg/kg) were i.p. administered. Saline was administered for the virus control group. The treatment was continued once a day for 7 consecutive days. The mice were monitored daily for weight and mortality until 21 d.p.i. The survival rate was processed using the log-rank test.
Another three groups of 1-day-old ICR suckling mice (11 newborn mice per group) were infected with 25 µL of EV-A71 H strain virus (10 6 PFU per mouse) intraperitoneally. After 4 h post infection, EIDD-1931 (200 mg/kg), EIDD-2801 (200 mg/kg), and saline were i.p. administered. The treatment was continued daily for 4 days. The mice were then euthanized, and the brain, heart, liver, intestines, lung, and limb muscles were separately harvested. The levels of viral RNA in each tissue were measured by qRT-PCR.

Ethics Statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Beijing Institute of Pharmacology and Toxicology. All work with infectious viruses was performed at the biosafety level 2 (BSL-2) or animal biosafety level 2 laboratory (ABSL-2).

Statistical Analysis
Statistical analyses were carried out using the GraphPad Prism 7.0 software. A logrank test was performed for the survival analysis. An unpaired two-tailed Student's t test, or a one-way analysis of variance was used to analyze the statistical significance of two or multiple groups, respectively. For each test, p < 0.05 was considered to indicate a statistically significant difference.

EIDD-1931 and EIDD-2801 Inhibit EV-A71 Infection In Vitro
To determine the inhibitory activity of EIDD-1931 and EIDD-2801 against EV-A71 virus, a cytopathic effect (CPE) protection assay was carried out using different cell lines. As shown in Figure 1B  To further explore the inhibitory efficacy of EIDD-2801 and EIDD-1931 on viral RNA replication and infectious viral particle generation, a qRT-PCR assay and a PFU assay were conducted, respectively. Treatment with EIDD-1931 or EIDD-2801 inhibited the replication of viral RNA in a dose-dependent manner ( Figure 2B,D), and the propagation of infectious viral particles was also inhibited (Figure 2A,C). EIDD-1931 seems to be 10 times more potent than EIDD-2801, which is in accordance with the EC 50 results.

EIDD-1931 and EIDD-2801 Reduce the Production of EV-A71 Virus Protein
To explore the potential of EIDD-1931 and EIDD-2801 for inhibiting EV-A71 virus protein production, we detected viral capsid protein (VP1) levels using immunofluorescence staining and Western blot assays. Fluorescence imaging showed that EIDD-1931 and EIDD-2801 strongly inhibited the production of VP1 proteins. As shown in Figure 2E, the expression of EV-A71 virus VP1 protein was almost completely blocked by EIDD-1931 at a concentration of 10 µM and partly inhibited by EIDD-2801 at a concentration of 100 µM, which is in accordance with the observations in the CPE inhibition and viral yield reduction assays. The results of Western blot revealed that the expression levels of viral VP1 proteins were significantly attenuated in the presence of EIDD-1931 or EIDD-2801 in a dose-dependent manner ( Figure 2F-I). Taken together, these data indicated that EIDD-1931 and EIDD-2801 can significantly reduce the expression of viral VP1 protein.

EIDD-1931 Acts at the Post-Entry Stage of EV-A71 infection
As a nucleoside analog, EIDD-1931 has been reported to be incorporated into nascent viral RNAs in place of cytidine, followed by an increasing frequency of lethal mutagenesis and a reduction in the generation of offspring virus. To determine which stage of the EV-A71 life cycle was affected by EIDD-1931, we performed a time-of-addition assay via incubation with the test compound at different time intervals according to the illustration in Figure 3A. EIDD-1931 and NITD008 belong to nucleoside analogue inhibitors, which have similar antiviral mechanism. NITD008 is not only a classical inhibitor against flavivirus infection, but also has potent anti-EV-A71 activity as reported in multiple literatures [33][34][35]. Therefore, we used it as the positive control drug for viral suppression. As expected, EIDD-1931 inhibited EV-A71 viral RNA production at stages III and IV, which indicates that EIDD-1931 acts at the post-entry stage of virus infection. No inhibition effects were observed at the pre-incubation, attachment, or internalization phases of the viral life cycle ( Figure 3B). To sum up, these data suggested that EIDD-1931 acts at the post-entry step.

EIDD-1931 and EIDD-2801 Protected 1-Day-Old ICR Suckling Mice from Lethal EV-A71 Challenge
To test the in vivo therapeutic efficacy of EIDD-1931 and EIDD-2801 against EV-A71 infection, we used a lethal 1-day-old ICR suckling mouse model for evaluation of antiviral activity [36]. After the mice were intraperitoneally (i.

EIDD-1931 and EIDD-2801 Protected 1-Day-Old ICR Suckling Mice from Lethal EV-A71 Challenge
To test the in vivo therapeutic efficacy of EIDD-1931 and EIDD-2801 against EV-A71 infection, we used a lethal 1-day-old ICR suckling mouse model for evaluation of antiviral activity [36]. After the mice were intraperitoneally (i.p.) infected with 10 6     Data for B and D are presented as means ± standard deviations. Survival data were analyzed with a log-rank test. ** p < 0.01, **** p < 0.0001.

EIDD-1931 and EIDD-2801 Reduce the Viral Loads in Various Tissues of EV-A71-Infected Mice
To further characterize the in vivo antiviral effects of EIDD-1931 and EIDD-2801, we determined the viral loads of various tissues in EV-A71-infected mice treated with the test compounds or vehicle. After the mice were infected with 106 PFU of EV-A71 virus (H strain), EIDD-1931 and EIDD-2801 at a dosage of 200 mg/kg or vehicle were administered via i.p. for 4 consecutive days. Then, the mice were dissected and several tissues, including brain, heart, intestine, liver, limb muscle, and lung, were collected to determine the viral RNA load using qRT-PCR. As shown in Figure 5, treatment with EIDD-1931 and EIDD-2801 significantly reduced the viral loads in the above tissues. These results suggested that EIDD-1931 and EIDD-2801 potently inhibit the viral replication of EV-A71 in mouse models. Then, the mice were euthanized, and brain (A), heart (B), intestine (C), liver (D), limb muscles (E) and lung (F) were separately harvested to determine the viral RNA loads using qRT-PCR. Data are presented as means ± standard deviations, and Student's unpaired t test was performed for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Discussion
Enteroviruses are diverse and spread easily. The epidemics and outbreaks of diseases caused by enteroviruses represent one of the most important public health problems all over the world that seriously threatens the health of children. Hence, effective antiviral treatment against these viruses is urgently needed.
As a key virus-encoded enzyme in the viral replication cycle, RNA-dependent RNA polymerase (RdRp) plays an important role in viral genome replication. Considering the similar structure of RdRps in different viruses and the conservation of their active elements, RdRps have become one of the best targets for developing broad-spectrum antiviral agents. EIDD-1931, as a ribonucleotide analog, has been established as an active agent against various RNA viruses in vitro. An in vivo activity study demonstrated that EIDD-1931 can be efficacious against respiratory syncytial virus and both seasonal and highly pathogenic avian influenza A virus in mouse models, reducing lung virus loads and alleviating disease biomarkers via oral (p.o.) administration [20]. Although EIDD-1931 exhibits good oral bioavailability in rodents, some researchers found that the plasma concentrations were low in cynomolgus macaques, which reflects poor bioavailability in humans. To overcome this problem, molnupiravir (EIDD-2801, the pro-drug of EIDD-1931) was designed. The data for EIDD-2801 showed that it has increased oral bioavailability in non-human primates and ferrets compared to EIDD-1931, but both have similar oral bioavailability in mice [24].
In this study, we reported that EIDD-1931 and EIDD-2801 have broad-spectrum antienterovirus potential in vitro. Further research on the mechanism of EIDD-1931 found that it acts at the post-entry step. However, the post-entry step includes virus replication, virus assembly, and virus release. EIDD-1931, as a ribonucleotide analog, can introduce lethal mutagenesis during viral RNA replication [19]. We speculate that EIDD-1931 acts at the replication stage of EV-A71. To verify this possibility, additional replicon experiments need to be performed. Because the enterovirus mainly infects infants and young children, we developed and tested a corresponding 1-day-old suckling mouse model. The in vivo data suggested that EIDD-1931 and EIDD-2801 have therapeutic significance in lethal EV-A71infected mice; when administered therapeutically for 4 consecutive days, they significantly reduced the viral load in various tissues of EV-A7-infected mice. The pharmacokinetic profile is an important reference for estimating clinical efficacy. The Cmax of EIDD-2801 was 16.13 µM following oral administration of a dose of 800 mg twice daily for 5 days, which is greater than the EC 50 of enteroviruses (the in vivo metabolite of EIDD-2801 was EIDD-1931), indicating that EIDD-2801 is a potential countermeasure against enteroviruses [37].
In conclusion, EIDD-1931 and EIDD-2801 exhibited powerful anti-enterovirus effects in vitro and in vivo and demonstrated excellent broad-spectrum anti-enterovirus activity in this study. Combined with complete in vivo data and the high safety of EIDD-2801, we believe that EIDD-2801 and its active form, EIDD-1931, may be a promising drug candidate against enterovirus infections. Although molnupiravir has obtained emergency authorization to treat SARS-CoV-2 infection in many countries, it is currently only approved for young adults over 18 years old. The enterovirus mainly infects children under 5 years old. Therefore, the potential risk of clinical application of molnupiravir in the treatment of enterovirus infection needs to be further studied.